Abstract
Published online ahead of print on 8 October 2004 as DOI 10.1099/ijs.0.03030-0.
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains CMS 39T and CMS 56T are AJ584832 and AJ584833, respectively.
Tables giving additional data relating to fatty acid profiles, phospholipids, 16S rRNA gene sequence similarity and DNADNA relatedness are available as supplementary material in IJSEM Online.
Juni & Heym (1986) described the genus Psychrobacter to accommodate Gram-negative, cocci or rod-shaped bacteria that lacked motility and possessed strictly oxidative metabolism. The genus belongs to the family Moraxellaceae, which includes two other genera, Moraxella and Acinetobacter (Bowman et al., 1996; Rossau et al., 1991). At the time of writing, the genus Psychrobacter comprises 15 species (Juni & Heym, 1986; Bowman et al., 1996, 1997; Maruyama et al., 2000; Denner et al., 2001; Romanenko et al., 2002; Yoon et al., 2003; Kämpfer et al., 2002; Vela et al., 2003; Bozal et al., 2003; Yumoto et al., 2003). Members of the genus Psychrobacter are known to be obligate or facultative psychrophiles and have been isolated from a wide range of habitats, including food, clinical samples, skin, gills and intestines of fish, sea water, penguin colonies in Antarctica, Antarctic sea ice and Japan Trench sediments (Juni, 1991; Bowman et al., 1996, 1997; Maruyama et al., 2000; Yumoto et al., 2003). We report here on two novel Psychrobacter species isolated from cyanobacterial mat samples collected from a pond, a lake and a stream in the McMurdo Dry Valley region of Antarctica.
Source of the organisms, media, growth conditions, morphology and biochemical characteristics
Twelve bacterial colonies were isolated to purity from cyanobacterial mat samples collected at three different localities in Antarctica: L3 pond in the Wright valley (strains CMS 27, CMS 28, CMS 29, CMS 30, CMS 31 and CMS 32), Adams glacier stream, Miers valley (strain CMS 39T), and lake Canopus (strains CMS 51, CMS 52, CMS 56T, CMS 58 and CMS 59) (Matsumoto, 1993; Matsumoto et al., 1993) using Antarctic bacterial medium (ABM) agar [0·5 % (w/v) peptone, 0·2 % (w/v) yeast extract and 1·5 % (w/v) agar, pH 7·0] as described by Reddy et al. (2000). Morphological and growth characteristics were determined as described by Reddy et al. (2000). For biochemical tests (as listed in Table 1 and as described under the species descriptions) the cultures were grown at 22 °C on ABM and tests were performed as described by Lanyi (1987) and Smibert & Krieg (1994). The ability of the cultures to utilize a carbon compound as the sole carbon source was investigated by supplementing minimal medium [1·05 % (w/v) K2HPO4, 0·45 % (w/v) KH2PO4, 0·1 % (w/v) (NH4)2SO4 and 1·5 % (w/v) agar)] with 0·5 % (w/v) of the filter-sterilized carbon compound. The sensitivity of the cultures to different antibiotics was checked using antibiotic discs supplied by HiMedia pvt. Ltd.
Table 1. Phenotypic characteristics of the 12 Psychrobacter isolates from Antarctica All 12 isolates are coccoid, non-motile and Gram-negative. All strains are positive for catalase, oxidase, lipase, phosphatase and nitrate reduction, but negative for H2S production, indole production, methyl red test, VogesProskauer reaction, citrate utilization, hydrolysis of starch, gelatin, aesculin and arginine, arginine decarboxylase and lysine decarboxylase. Growth observed at pH 9 but not at pH 5. Growth observed at 430 °C but not at 37 °C and can tolerate 7·5 % NaCl. Utilize L-asparagine and L-glutamate but not D-fructose, D-mannose, sorbitol, lactose, sucrose, D-xylose, L-arabinose, inulin, dulcitol, trehalose, D-maltose, adonitol, D-melibiose, melizitol, D-mannitol, dextran, methanol, xanthine, butanol, hexadecane, L-leucine, L-isoleucine, L-methionine, L-valine, threonine, L-histidine, L-tryptophan or L-cysteine as the sole carbon source. Do not produce acid from L-arabinose, lactose or sucrose. Sensitive to chloramphenicol (30 µg), gentamicin (10 µg), polymyxin B (300 µg), nalidixic acid (30 µg), tetracycline (30 µg), tobramycin (20 µg) and rifampicin (5 µg) and resistant to lincomycin (2 µg). +, Positive; , negative; S, sensitive; R, resistant.
All 12 isolates are Gram-negative, non-motile, coccoid, psychrophilic and halotolerant (up to 8 % NaCl) bacteria and possess many similar morphological and biochemical characteristics (Table 1). However, they could be differentiated based on their ability to hydrolyse urea, to tolerate 12 % NaCl, to utilize phenylalanine, alanine and tyrosine as sole carbon sources and to produce acid from glucose and in their sensitivity to ten different antibiotics (Table 1). Chemotaxonomic grouping by SDS-PAGE analysis (Laemmli, 1970) shows that the 12 isolates can be divided into three groups (data not shown): Group I (strains CMS 27, CMS 28, CMS 29, CMS 30, CMS 31, CMS 32), Group II (strain CMS 39T) and Group III (strains CMS 51, CMS 52, CMS 56T, CMS 58 and CMS 59). Isolates of the same group exhibited identical protein profiles and very similar phenotypic characteristics (Table 1).
Chemotaxonomic characteristics and phylogenetic analysis
Based on the phenotypic characteristics listed in Table 1, the presence of C16 : 1ω7c, C17 : 1ω8c and C18 : 1ω9c as the major fatty acids (Table A, available as supplementary material in IJSEM Online), DNA G+C content in the range 4146 % (Table 1) and 16S rRNA gene sequence similarity ranging from 96·8 to 99·1 % (Table B, available as supplementary material in IJSEM Online) with recognized species of Psychrobacter, it appears that the 12 isolates are related to members of the genus Psychrobacter. The six isolates in Group I, which had similar phenotypic characteristics (Table 1) and identical 16S rRNA gene sequences (CMS 27, GenBank/EMBL/DDBJ accession no. AJ748266; CMS 28, AJ748267; CMS 29, AJ748268; CMS 30, AJ584831; CMS 31, AJ748269; CMS 32, AJ748270), probably represented the same species and were closely related to Psychrobacter okhotskensis MD17T based on data from 16S rRNA gene sequence similarity (98·6 %) and DNADNA relatedness analysis (72 %) (Table B). Strain CMS 30, a representative strain of Group I, shares similar phenotypic characteristics (Table 2) with P. okhotskensis MD17T and differs only in a few physiological traits (Table 2) and in its polar lipid profile (Table C, available as supplementary material in IJSEM Online). Therefore, the six isolates in Group I are identified as strains representing P. okhotskensis. All the above phenotypic and chemotaxonomic characteristics and 16S rRNA gene sequence analysis were determined based on standard procedures as described by Shivaji et al. (1989, 2000) and Reddy et al. (2002a, 2003). The 16S rRNA gene was amplified and sequenced (Lane, 1991; Shivaji et al., 2000), and the sequences were aligned using CLUSTAL W (Thompson et al., 1994) and subjected to phylogenetic analysis using the PHYLIP program (Felsenstein, 1993). Evolutionary distances were calculated using Kimura's two-parameter model (Kimura, 1980) using the DNADIST program of PHYLIP (Felsenstein, 1993).
Table 2. Phenotypic characteristics that differentiate Psychrobacter okhotskensis CMS 30, Psychrobacter vallis CMS 39T and Psychrobacter aquaticus CMS 56T between themselves and recognized species of the genus Psychrobacter CMS 30, CMS 39T and CMS 56T have the following identical phenotypic characteristics: grow at 430 °C, tolerate 10 % NaCl and grow optimally at pH 7. The three species are positive for catalase, oxidase, lipase and alkaline phosphatase and reduce nitrate to nitrite. Negative for gelatinase, arginine dihydrolase, arginine decarboxylase, lysine decarboxylase, indole production and VogesProskauer test and do not hydrolyse aesculin, starch or cellulose. Utilize trehalose and L-glutamic acid as sole carbon source but not L-arabinose, D-cellobiose, citrate, dextran, dulcitol, D-fructose, D-galactose, inulin, lactose, D-mannose, D-mannitol, D-maltose, melizitose, D-melibiose, D-raffinose, L-rhamnose, D-sorbose, sorbitol, sucrose, succinic acid, thioglycolate, D-xylose, L-alanine, L-aspartic acid, cysteine, L-glycine, L-histidine, L-leucine, L-isoleucine, L-lysine, L-methionine, L-proline, L-tryptophan or threonine. Do not produce acid or gas from L-arabinose, D-fructose, D-galactose, D-maltose, D-melibiose or lactose. The three isolates are sensitive to ampicillin (10 µg), amoxicillin (100 µg), chloramphenicol (30 µg), co-trimoxazole (25 µg), gentamicin (10 µg), nalidixic acid (30 µg), nystatin (100 µg), polymyxin B (300 µg), rifampicin (5 µg), streptomycin (10 µg), tetracycline (30 µg) and tobramycin (20 µg) and resistant to lincomycin (2 µg). Ubiquinone-8, phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol are present. The predominant fatty acid is C18 : 1ω9c. Taxa: 1, P. okhotskensis (CMS 30); 2, P. vallis (CMS 39T); 3, P. aquaticus (CMS 56T); 4, P. luti (LMG 21276T)1; 5, P. marincola (DSM 14160T)2; 6, P. submarinus (DSM 14161T)2; 7, P. proteolyticus (DSM 13887T)3; 8, P. faecalis (DSM 14664T)4; 9, P. jeotgali (YKJ-103T)5; 10, P. frigidicola (ACAM 304T)6; 11, P. glacincola (ATCC 700754T)6; 12, P. urativorans (ATCC 15174T)7; 13, P. immobilis (DSM 7229T)7; 14, P. pacificensis (IFO 16270T)8; 15, P. phenylpyruvicus (ATCC 23333T)6; 16, P. fozii (LMG 21280T)1; 17, P. okhotskensis (JCM 11840T)9; 18, P. pulmonis (CECT 5989T)10. Data from: 1Bozal et al. (2003), 2Romanenko et al. (2002), 3Denner et al. (2001), 4Kämpfer et al. (2002), 5Yoon et al. (2003), 6Bowman et al. (1996, 1997), 7Juni & Heym (1986), 8Maruyama et al. (2000), 9present study and 10Vela et al. (2003). +, Positive; , negative; ND, not done; V, variable; W+, weak positive.
Strains CMS 39T (Group II) and CMS 56T (as a representative of Group III) also possess characteristics similar to those of members of the genus Psychrobacter (Tables 1 and 2, and Tables A and B). Riboprinting was carried out to establish the similarity of the present isolates between themselves and recognized species of Psychrobacter according to the protocol recommended by the manufacturer of the Riboprinting System (DuPont Qualicon). The results clearly demonstrated that strains CMS 30, CMS 39T and CMS 56T are different and that their riboprints do not match with those of recognized Psychrobacter species (Fig. 1).
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16S rRNA gene sequence analysis indicated that strains CMS 30 (as a representative of Group I), CMS 39T and CMS 56T are closely related to each other (99·499·9 %) and to recognized type strains of species of the genus Psychrobacter (96·899·1 %) (Table B and Fig. 2). However, the three strains are more closely related to Psychrobacter luti LMG 21276T with a similarity of 99 % (Table B and Fig. 1). The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains for Groups II and III are as follows: CMS 39T, AJ584832; CMS 51, AJ830004; CMS 52, AJ830005; CMS 56T, AJ584833; CMS 58, AJ830006; CMS 59, AJ830007. Strains CMS 39T and CMS 56T could be differentiated from P. luti LMG 21276T in that they tolerated higher NaCl concentrations, and could utilize glycerol and trehalose but not citrate, asparagine, histidine or proline as sole carbon sources (Table B). DNADNA hybridization was performed by the membrane filter method of Reddy et al. (2002b) and the results indicate that CMS 39T and CMS 56T share a DNADNA relatedness of only 25 % between themselves and of 34 and 10 %, respectively, with P. luti LMG 21276T, indicating that the two isolates are distinctly different from P. luti LMG 21276T (Table B). Strains CMS 39T and CMS 56T can also be differentiated from the other closely related species of the genus Psychrobacter in that they varied in their riboprint profiles (Fig. 2), in their phenotypic characteristics (Table 2) and in their fatty acid composition (Tables C and D). Furthermore, CMS 39T and CMS 56T exhibited less than 70 % DNADNA relatedness with 16 of the 17 species that exhibited greater than 97 % 16S rRNA gene sequence similarity (Table B). Therefore, it is proposed to assign strains CMS 39T and CMS 56T to two novel species, for which the names Psychrobacter vallis sp. nov. and Psychrobacter aquaticus sp. nov., respectively, are proposed.
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Description of Psychrobacter vallis sp. nov.
Psychrobacter vallis [val'lis. L. n. vallis (or valles) a valley, vale; L. gen. n. vallis of a valley].
On Zobell Marine 2296 agar, forms circular, convex, smooth and opaque colonies with a diameter of 23 mm. Cells are Gram-negative, non-motile, coccoid and psychrophilic (growth observed at 430 °C). Negative for urease. Utilizes erythritol, D-glucose, inositol, pyruvate, D-ribose, L-arginine, L-glutamine, L-phenylalanine, L-serine and L-tyrosine as sole carbon source but not acetate, fumaric acid, hydroxybutyric acid, lactic acid, L-asparagine or L-valine. Produces acid from D-glucose but not from D-mannose. Sensitive to chlortetracycline (30 µg) and trimethoprim (5 µg). Resistant to bacitracin (10 µg), carbenicillin (50 µg), colistin (10 µg), erythromycin (15 µg), furazolidone (50 µg), furoxone (100 µg), kanamycin (30 µg) and nitrofurantoin (300 µg). Table B (available in IJSEM Online) lists phenotypic characteristics common to the two novel species. Fatty acids present include C10 : 0 (1·1 %), C16 : 0 (1·4 %), C17 : 0 (0·2 %), C18 : 0 (0·9 %), iso-C16 : 0 (0·2 %), C14 : 1 (0·2 %), C16 : 1ω7c (19·5 %), C17 : 1ω8c (9·3 %) and C18 : 1ω9c (65·7 %). Ubiquinone-8 is the respiratory quinone and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol are the major polar lipids present.
The type strain is CMS 39T (=DSM 15337T=MTCC 4208T).
Description of Psychrobacter aquaticus sp. nov.
Psychrobacter aquaticus (a.qua.ti'cus. L. masc. adj. aquaticus living, growing or found in or by the water, aquatic).
On Zobell Marine 2296 agar, forms circular, convex, smooth and opaque colonies with a diameter of 23 mm. Cells are Gram-negative, non-motile, coccoid and psychrophilic (growth observed at 430 °C). Positive for urease and negative for β-galactosidase. Utilizes L-phenylalanine as sole carbon source but not acetate, cellulose, erythritol, fumaric acid, glucose, meso-inositol, lactic acid, pyruvate, D-ribose, L-arginine, L-asparagine, L-glutamine, L-serine, L-tyrosine or L-valine. Produces acid from D-mannose but does not produce acid or gas from D-glucose. Sensitive to bacitracin (10 µg), carbenicillin (50 µg), chlortetracycline (30 µg), erythromycin (15 µg), nitrofurantoin (300 µg), nitrofurazone (10 µg), oxytetracycline (30 µg), penicillin (10 µg) and trimethoprim (5 µg). Resistant to colistin (10 µg), furazolidone (50 µg), furoxone (100 µg) and kanamycin (30 µg). Table B (available in IJSEM Online) lists phenotypic characteristics common to the two novel species. The fatty acids present are common to all the strains of the species and vary quantitatively as follows: C10 : 0 (0·32 %), C12 : 0 (0·10·4 %), C16 : 0 (0·32·4 %), C18 : 0 (0·51·2 %), iso-C16 : 0 (0·10·4 %), C14 : 1 (0·51·3 %), C16 : 1ω7c (12·423 %), C17 : 1ω8c (11·220 %) and C18 : 1ω9c (5864·3 %). Ubiquinone-8 is the respiratory quinone and phosphatidylethanolamine, phosphatidylglycerol and diphosphatidylglycerol are the major polar lipids present.
The type strain is CMS 56T (=DSM 15339T=MTCC 4386T).
Acknowledgements
This work was supported by a grant from the Department of Biotechnology, Government of India and Indo-French Centre for Promotion of Advanced Research, New Delhi, India. The riboprinting work was carried out at the DSMZ, Germany, through the UNESCO-IUMS-MIRCENS-SGM short-term fellowship awarded to G. S. N. R. We thank Dr J. S. S. Prakash and Dr S. Dube for their help with fatty acid analysis.References
Bowman, J. P., Nichols, D. S. & McMeekin, T. A. (1997). Psychrobacter glacincola sp. nov., a halotolerant, psychrophilic bacterium isolated from Antarctic sea ice. Syst Appl Microbiol 20, 209215.
Bozal, N., Montes, M. J., Tudela, E. & Guinea, J. (2003). Characterization of several Psychrobacter strains isolated from Antarctic environments and description of Psychrobacter luti sp. nov. and Psychrobacter fozii sp. nov. Int J Syst Evol Microbiol 53, 10931100.
Denner, E. B. M., Mark, B., Busse, H.-J., Turkiewicz, M. & Lubitz, W. (2001). Psychrobacter proteolyticus sp. nov., a psychrophilic, halotolerant bacterium isolated from the Antarctic krill Euphausia superba Dana, excreting a cold-adapted metalloprotease. Syst Appl Microbiol 24, 4453.[CrossRef][Medline]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.5c. Department of Genetics, University of Washington, Seattle, USA.
Juni, E. (1991). The genus Psychrobacter. In The Prokaryotes, pp. 32413246. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K. H. Schleifer. New York: Springer.
Juni, E. & Heym, G. A. (1986). Psychrobacter immobilis gen. nov., sp. nov.: genospecies composed of gram-negative, aerobic, oxidase-positive coccobacilli. Int J Syst Bacteriol 46, 388391.
Kämpfer, P., Albrecht, A., Buczolits, S. & Busse, H.-J. (2002). Psychrobacter faecalis sp. nov., a new species from a bioaerosol originating from pigeon faeces. Syst Appl Microbiol 25, 3136.[CrossRef][Medline]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111120.[CrossRef][Medline]
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[CrossRef][Medline]
Lane, D. J. (1991). 16S/23S rRNA sequencing. In Nucleic Acid Techniques in Bacterial Systematics, pp. 115147. Edited by E. Stackebrandt & M. Goodfellow. New York: Wiley.
Lanyi, B. (1987). Classical and rapid identification methods for medically important Bacteria. Methods Microbiol 19, 167.
Maruyama, A., Honda, D., Yamamoto, H., Kitamura, K. & Higashihara, T. (2000). Phylogenetic analysis of psychrophilic bacteria isolated from the Japan Trench, including a description of the deep sea species Psychrobacter pacificensis sp. nov. Int J Syst Evol Microbiol 50, 835846.[Abstract]
Matsumoto, G. I. (1993). Geochemical features of the McMurdo Dry Valley lakes, Antarctica. Physical and biogeochemical processes in Antarctic Lakes. Antarct Res Ser 49, 95118.
Matsumoto, G. I., Ohtani, S. & Hiroto, K. (1993). Biogeochemical features of hydrocarbons in cyanobacterial mats from the McMurdo Dry Valley, Antarctica. Proc NIPR Symp Polar Biol 6, 98105.
Reddy, G. S. N., Aggarwal, R. K., Matsumoto, G. I. & Shivaji, S. (2000). Arthrobacter flavus sp. nov., a psychrophilic bacterium isolated from a pond in McMurdo Dry Valley, Antarctica. Int J Syst Evol Microbiol 50, 15531561.[Abstract]
Reddy, G. S. N., Prakash, J. S. S., Matsumoto, G. I., Stackebrandt, E. & Shivaji, S. (2002a). Arthrobacter roseus sp. nov., a psychrotolerant bacterium isolated from an Antarctic cyanobacterial mat sample. Int J Syst Evol Microbiol 52, 10171021.[Abstract]
Reddy, G. S. N., Prakash, J. S. S., Vairamani, M., Prabhakar, S., Matsumoto, G. I. & Shivaji, S. (2002b). Planococcus antarcticus and Planococcus psychrophilus sp. nov. isolated from cyanobacterial mat samples collected from ponds in Antarctica. Extremophiles 6, 253261.[CrossRef][Medline]
Reddy, G. S. N., Raghavan, P. U. M., Sarita, N. B., Prakash, J. S. S., Nagesh, N., Delille, D. & Shivaji, S. (2003). Halomonas glaciei sp. nov. isolated from fast ice of Adelie Land, Antarctica. Extremophiles 7, 5561.[Medline]
Romanenko, L. A., Schumann, P., Rhode, M., Lysenko, A. M., Mikhailov, V. V. & Stackebrandt, E. (2002). Psychrobacter submarinus sp. nov. and Psychrobacter marincola sp. nov., psychrophilic halophiles isolated from marine environments. Int J Syst Evol Microbiol 52, 12911297.[Abstract]
Rossau, R., van Landschoot, A., Gillis, M. & De Ley, J. (1991). Taxonomy of Moraxellaceae fam. nov., a new bacterial family to accommodate the genera Moraxella, Acinetobacter, and Psychrobacter and related organisms. Int J Syst Bacteriol 41, 310319.
Shivaji, S., Rao, N. S., Saisree, L., Sheth, V., Reddy, G. S. N. & Bhargava, P. M. (1989). Isolation and identification of Pseudomonas spp. from Schirmacher Oasis, Antarctica. Appl Environ Microbiol 55, 767770.
Shivaji, S., Bhanu, N. V. & Aggarwal, R. K. (2000). Identification of Yersinia pestis as the causative organism of plague in India as determined by 16S rDNA sequencing and RAPD-based genomic fingerprinting. FEMS Microbiol Lett 189, 247252.[CrossRef][Medline]
Smibert, R. M. & Krieg, N. R. (1994). Phenotypic characterization. In Methods for General and Molecular Bacteriology, pp. 607654. Edited by P. Gerhardt. Washington, DC: American Society for Microbiology.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 46734680.
Vela, A. I., Collins, M. D., Latre, M. V., Mateos, A., Moreno, M. A., Hutson, R., Dominguez, L. & Fernandez-Garayzabal, J. F. (2003). Psychrobacter pulmonis sp. nov., isolated from the lungs of lambs. Int J Syst Evol Microbiol 53, 415419.
Yoon, J.-H., Kang, K. H. & Park, Y.-H. (2003). Psychrobacter jeotgali sp. nov., isolated from jeotgal, a traditional Korean fermented seafood. Int J Syst Evol Microbiol 53, 449454.
Yumoto, I., Hirota, K., Sogabe, Y., Nodasaka, Y., Yokota, Y. & Hoshino, T. (2003). Psychrobacter okhotskensis sp. nov., a lipase-producing facultative psychrophile isolated from the coast of the Okhotsk Sea. Int J Syst Evol Microbiol 53, 19851989.